1. Field of the Invention
The present invention relates generally to instrumentation and methods for manipulating and studying electrical properties of epithelial cells, intact biological membranes, and tissues.
2. Description of the Related Art
The Ussing chamber is named after Hans H. Ussing, who pioneered the concept of measuring ion flux across epithelial tissues via electrical measurements in the 1950s. See Ussing, H. H. & Zerahn, K. (1951) Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. Scand. 23: 110-127, hereby expressly incorporated by reference in its entirety.
Ussing's original studies used intact frog skin, but over the years, the Ussing chamber has become a preferred tool to study transport across a variety of epithelial cells, intact biological membranes, and tissues. More recently, progress has been made in the ability to grow primary epithelial cells or immortalized cell lines on a porous supporting membrane. Under appropriate culture conditions, these cells grow to confluence, establish polarity, and can form tight junctions between cells, creating a high resistance monolayer (ca. ≧0.3 kohm/cm2) suitable for transepithelial measurements. The ability to use primary cells or engineered cell lines allows the biophysical and pharmacological study of epithelial function, including effects on ion channels or transporters. Despite its utility and diverse applications, the experiments remain laborious and time-consuming. This limits the utility of this technique for modern research methods, including screening of molecules or proteins for effects on ion transport.
A typical Ussing chamber is shown in FIG. 1. As shown, this Ussing chamber consists of three main parts: a first compartment, a second compartment, and a middle insert that carries the membrane on which the cell layer resides. The first compartment 10 is separated from a second compartment 12 by the middle insert 14. The middle insert 14 contains a membrane support 16 on which a confluent epithelial cell layer 18 has been grown. The cells of the cell layer are held together by tight junctions. The cell layer effectively prevents molecules from traveling between the first and second compartments unless such a molecule passes through one of the cells by entering through a cellular channel located on side of the cell and then exiting the cell through a cellular channel located on the other side of the cell. Often, one ionic component of the salt in one compartment is higher than in the other and the ionic flux down the concentration gradient is measured, although this is not required. This flux provides information on the channels or transporters in the cell membrane. In this example, the first compartment has a higher KCl concentration than the second compartment. A chloride ion flux is thus produced by chloride ions passing through the cells of the cell layer 18.
FIG. 2 shows the use of a voltage clamp to help measure this flux. In this specific example, as Cl− ions move down the concentration gradient, the potential becomes more negative in the second compartment 12. This potential change is sensed by voltage electrodes 20 and used by the servo loop to command a charge injection via the charge injection or current electrodes 22. In this way, the potential change is “short-circuited” and the voltage across the cell layer remains “clamped” at a constant level. The amount of charge injected is equal to the amount of Cl− that moves across the cell layer, which allows the Cl− flux to be measured. The electronics responsible for pumping this charge can also report it to an external data acquisition system. Both voltage and current electrodes in this arrangement are silver/silver chloride (Ag/AgCl) encased in plastic pipettes 24 filled with KCl/agar 26 (10% agar in 1 M KCl). Such compound electrodes are advantageous because sometimes the chloride concentrations in one or both compartments are modified during the experiment by addition of reagents or solutions. The KCl/agar provides a constant Cl− environment surrounding the Ag/AgCl so that chloride concentrations changes in the bath do not cause voltage jumps. In the current state of the art, the voltage clamping electronics are typically fitted with a manual user interface which includes a complicated assortment of knobs, switches, and dials through which the user enters all parameters needed to set up the experiment. As for the chamber itself, it is typically made out of machined and polished Plexiglas and its dimensions are usually about 3×6×7 cm. Typically, each compartment's volume is about 5 mL, but the minimum workable volume is about 3 mL. Cells can be grown on a Snapwell™ plate, which is available from Corning Costar (Cambridge, Mass.). A Snapwell™ plate typically contains six wells, each with a polycarbonate membrane support on which a cell layer can be grown. Once confluence is reached, one Snapwell™ support is removed and installed into the insert, which is then mounted between the two halves of the Ussing chamber. The area of the microporous membrane support on each Snapwell™ is typically about 1.1 cm2.
As described above, the typical Ussing chamber experiment is a time-consuming, cumbersome, and labor-intensive process which includes (1) zeroing the electrodes to compensate for the solution resistance, (2) mounting one Snapwell™ on the insert, (3) installing the insert into the chamber, (4) inserting the electrodes, (5) adding solutions and reagents, (6) manipulating the electronics manual interface, and (7) collecting the data. Silver/silver chloride electrodes also wear out, and rebuilding these compound electrodes usually involves a cumbersome process of handling melted agar. A typical Ussing experiment takes several hours, yet provides only one data set, as only one Snapwell™ can be tested at a time. In the context of drug screening, where it is often desirable to screen hundreds or thousands of compounds, such throughput is unacceptably low. Even in the scenario of a secondary screen, or the profiling of medicinal chemistry compounds, this throughput of one data point in several hours is still too low to satisfy the need to test a number of compounds at various concentrations in order to calculate an effective concentration, for example, when obtaining a dose response profile. What is needed in the art is a Ussing chamber apparatus and method for its use that allows greater throughput.